Determining analyte concentration from variant concentration distribution in measurable species

ABSTRACT

A biosensor system determines an analyte concentration of a biological sample using an electrochemical process without Cottrell decay. The biosensor system generates an output signal having a transient decay, where the output signal is not inversely proportional to the square root of the time. The transient decay is greater or less than the −0.5 decay constant of a Cottrell decay. The transient decay may result from a relatively short incubation period, relatively small sample reservoir volumes, relatively small distances between electrode surfaces and the lid of the sensor strip, and/or relatively short excitations in relation to the average initial thickness of the reagent layer. The biosensor system determines the analyte concentration from the output signal having a transient decay.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Nonprovisionalapplication Ser. No. 13/904,208, filed May 29, 2013, titled “TransientDecay Amperometry Biosensors,” which is a divisional of U.S.Nonprovisional application Ser. No. 13/210,738, now issued as U.S. Pat.No. 8,470,604, filed Aug. 16, 2011, titled “Transient DecayAmperometry,” which is a continuation of U.S. Nonprovisional applicationSer. No. 11/875,942, now issued as U.S. Pat. No. 8,026,104, filed Oct.21, 2007, titled “Transient Decay Amperometry,” which claims the benefitof (1) U.S. Provisional Application No. 60/869,625, filed Dec. 12, 2006,titled “Transient Decay Amperometry,” (2) U.S. Provisional ApplicationNo. 60/869,557, filed Dec. 11, 2006, titled “Transient DecayAmperometry,” and (3) U.S. Provisional Application No. 60/854,060, filedOct. 24, 2006, titled “Transient Decay Amperometry,” all of which areincorporated by reference in their entirety.

BACKGROUND

Biosensors provide an analysis of a biological fluid, such as wholeblood, urine, or saliva. Typically, a biosensor analyzes a sample of thebiological fluid to determine the concentration of one or more analytes,such as alcohol, glucose, uric acid, lactate, cholesterol, or bilirubin,in the biological fluid. The analysis is useful in the diagnosis andtreatment of physiological abnormalities. For example, a diabeticindividual may use a biosensor to determine the glucose level in wholeblood for adjustments to diet and/or medication.

Biosensors may be implemented using bench-top, portable, and likemeasurement devices. The portable measurement devices may be hand-held.Biosensors may be designed to analyze one or more analytes and may usedifferent volumes of biological fluids. Some biosensors may analyze asingle drop of whole blood, such as from 0.25-15 microliters (μL) involume. Examples of portable measurement devices include the AscensiaBreeze® and Elite® meters of Bayer Corporation; the Precision®biosensors available from Abbott in Abbott Park, Ill.; Accucheck®biosensors available from Roche in Indianapolis, Ind.; and OneTouchUltra® biosensors available from Lifescan in Milpitas, Calif. Examplesof bench-top measurement devices include the BAS 100B Analyzer availablefrom BAS Instruments in West Lafayette, Ind.; the ElectrochemicalWorkstation available from CH Instruments in Austin, Tex.; anotherElectrochemical Workstation available from Cypress Systems in Lawrence,Kans.; and the EG&G Electrochemical Instrument available from PrincetonResearch Instruments in Princeton, N.J.

Biosensors usually measure an electrical signal to determine the analyteconcentration in a sample of the biological fluid. The analyte typicallyundergoes an oxidation/reduction or redox reaction when an input signalis applied to the sample. An enzyme or similar species may be added tothe sample to enhance the redox reaction. The input signal usually is anelectrical signal, such as a current or potential. The redox reactiongenerates an output signal in response to the input signal. The outputsignal usually is an electrical signal, such as a current or potential,which may be measured and correlated with the concentration of theanalyte in the biological fluid.

Many biosensors include a measurement device and a sensor strip. Thesensor strip may be adapted for use outside, inside, or partially insidea living organism. When used outside a living organism, a sample of thebiological fluid is introduced into a sample reservoir in the sensorstrip. The sensor strip may be placed in the measurement device before,after, or during the introduction of the sample for analysis. Wheninside or partially inside a living organism, the sensor strip may becontinually immersed in the sample or the sample may be intermittentlyintroduced to the strip. The sensor strip may include a reservoir thatpartially isolates a volume of the sample or be open to the sample.Similarly, the sample may continuously flow through the strip or beinterrupted for analysis.

The measurement device usually has electrical contacts that connect withthe electrical conductors of the sensor strip. The electrical conductorstypically connect to working, counter, and/or other electrodes thatextend into the sample reservoir. The measurement device applies theinput signal through the electrical contacts to the electricalconductors of the sensor strip. The electrical conductors convey theinput signal through the electrodes into the sample present in thesample reservoir. The redox reaction of the analyte generates an outputsignal in response to the input signal. The measurement devicedetermines the analyte concentration in response to the output signal.

The sensor strip may include reagents that react with the analyte in thesample of biological fluid. The reagents may include an ionizing agentto facilitate the redox reaction of the analyte, as well as mediators orother substances that assist in transferring electrons between theanalyte and the conductor. The ionizing agent may be an oxidoreductase,such as an analyte specific enzyme, which catalyzes the oxidation ofglucose in a whole blood sample. The reagents may include a binder thatholds the enzyme and mediator together.

Many biosensors use amperometric methods where an electrical signal ofconstant potential (voltage) is applied to the electrical conductors ofthe sensor strip while the measured output signal is a current. Thus, inan amperometric system current may be measured as a constant potentialis applied across the working and counter electrodes of the sensorstrip. The measured current then may be used to determine the presenceof and/or quantify the analyte in the sample. Amperometry measures therate at which the measurable species, and thus the analyte, is beingoxidized or reduced at the working electrode. In addition to analytes,biological substrates and mediators, for example, may serve asmeasurable species

As the time during which the input signal is applied to the sensor stripincreases, the rate at which the measurable species is oxidized orreduced at the working electrode decreases. Thus, after an initialperiod of high current output, the current recorded from the sensorstrip decreases as the input signal continues to be applied. Thiscurrent decrease with time may be referred to as an electrochemicaldecay, and the rate of this decay may be correlated with theconcentration of measurable species, and thus the analyte, in thesample. An electrochemical decay may be a transient or Cottrell decay.

The electrochemical decay may be correlated with the analyteconcentration in the sample by expressing the decay with an equationdescribing a line that relates current with time by the natural logfunction (ln), for example. Thus, the output current may be expressed asa function of time with an exponential coefficient, where negativeexponential coefficients indicate a decay process. After the initialdecrease in current output, the rate of decrease may remain relativelyconstant or continue to fluctuate.

U.S. Pat. No. 5,942,102 (“the '102 patent”) describes the relationshipbetween measured output current and time during a conventional analysis.An electrical signal is input to a sensor strip about 60 seconds afterintroduction of the whole blood sample to the strip. Initially, arapidly decreasing current is observed, which is followed by arelatively constant or “steady-state” current output that is generatedby the feedback of mediator from the counter to the working electrode.The feedback of the mediator provided by the short distance between theelectrodes results in the current becoming substantially independent oftime after the initial decrease. In this conventional analysis, theanalyte concentration of the sample may be determined from theconcentration and diffusion coefficient of the mediator as determinedby: (1) measuring current as a function of time; and then (2) estimatingthe steady state current.

While the analysis method described in the '102 patent relies on thesteady-state portion of the current decay, U.S. Pat. Nos. 6,153,069(“the '069 patent”) and U.S. Pat. Nos. 6,413,411 (“the '411 patent”)describe methods where the concentration of a mediator, and thus theunderlying analyte, is determined from the diffusion coefficient of themediator. These systems are configured to provide a rate of currentdecay that is described by the Cottrell equation.

Current measurements demonstrate Cottrell decay when the measuredcurrent is inversely proportional to the square root of time. Currentmeasurements with Cottrell decay may be described by the Cottrellequation given below as Equation (1):

$\begin{matrix}{{{i(t)} = {{{nFAC}^{b}\left( \frac{D}{\pi\; t} \right)}^{1/2} = {{{nFAC}^{b}\left( \frac{D}{\pi} \right)}^{1/2}t^{- 0.5}}}},} & (1)\end{matrix}$where i is the measured current; C^(b) is the bulk concentration of theelectrochemically active species in mol/cm³; A is the electrode area incm²; F is the Faraday constant of 96,500 coul/equivalent; n is thenumber of electrons transferred in equivalents/mol.; D is the diffusioncoefficient in cm²/sec; and t is the time of the electrochemicalreaction in seconds. Thus, the Cottrell equation describes current as anexponential function of time, having a decay constant or exponentialcoefficient of −0.5. Further details of the Cottrell equation and theboundary conditions required for Cottrell behavior may be found inchapter 5, pp. 136-45, of Electrochemical Methods: Fundamentals andApplications by Bard and Faulkner (1980).

A system designed to operate with a Cottrell current decay requires adecay constant of −0.5. An electrochemical system demonstrating a −0.5decay constant implies that the requirements of a Cottrell current arepresent, namely that the analyte has completely converted to ameasurable species and that a substantially constant concentrationdistribution of this measurable species occupies the sample reservoirbefore current measurement. These requirements are further described inthe '069 and '411 patents.

Column 4, Lines 39-40 of the '411 patent discloses that initialincubation periods of 15 to 90 seconds, preferably from 20 to 45seconds, are used for glucose testing. After the initial incubationperiod and application of a single excitation input signal, currentmeasurements demonstrating Cottrell decay may be recorded from 2 to 30seconds or preferably from 10 to 20 seconds following application of theinput signal to the sensor strip. The requirement of a longer initialincubation period also is depicted in FIG. 7 of the '411patent, wherethe sample was allowed to react in the sensor strip (incubate) for 160seconds before application of the input signal.

The longer incubation periods required to completely convert the analyteto measurable species provide: (1) time for hydration of the reagentlayer containing the reagents; and (2) time for the reagents to convertthe analyte. For example, column 4, lines 36-44 of the '411 patentdescribes an incubation period of sufficient length to allow theenzymatic reaction to reach completion. After this incubation period,where the glucose analyte is fully converted to a measurable species,the instrument imposes a known potential across the electrodes tomeasure the resulting diffusion limited (i.e. Cottrell) current atspecific times during the resulting Cottrell current decay. Thus, theconversion of the analyte to the measurable species is completed beforeCottrell decay is observed. Complete hydration of the reagent layer alsois recognized in the '411 patent as a requirement for Cottrell decay.The '411 patent discloses that incomplete wetting of the reagent resultsin a failure of the system to follow the Cottrell curve decay, whichresults in an inaccurate analyte concentration value being obtained.

In addition to an extended incubation period, Cottrell decay alsorequires a substantially constant concentration distribution of ameasurable species in the sample as the distance from the electrodesurface increases. A substantially constant concentration distributionmay be achieved with: (1) relatively large sample volumes; and/or (2) arelatively large distance between facing planar electrodes orsubstantially planar electrodes and the bottom surface of the sensorstrip lid. For example, column 8, line 40 of the '069 patent describes aworking electrode occupying a sample reservoir providing a 50 μL samplevolume where the vertical distance between the working electrode and thelid is from 500-2000 μm. In another example, unlike the closely spacedelectrodes of the '102 patent, the distance between the working andcounter electrodes described in column 7, lines 62-66 of the '411 patentmust be at least 100 microns, and preferably greater than 100 microns.

Conventional analysis methods typically lengthen the time required toanalyze samples by requiring incubation periods, electrode distances,and sample reservoir volumes sufficient to allow the system to haveCottrell decay. Accordingly, there is an ongoing need for improvedbiosensors; especially those that more quickly determine the analyteconcentration of a sample and do not rely on the estimation of a steadystate current value. The systems, devices, and methods of the presentinvention overcome at least one of the disadvantages associated withconventional biosensors.

SUMMARY

The present invention provides a biosensor system that determines ananalyte concentration of a biological sample from an output signalhaving a transient decay. The output signal is not inverselyproportional to the square root of the time, and thus has a decayconstant greater or less than the decay constant of a Cottrell decay.

In one aspect, a method for determining an analyte concentration in asample includes applying an input signal to the sample after anincubation period, generating an output signal having a transient decayin response to a redox reaction of a measurable species; and determiningthe analyte concentration from the output signal. The analyte mayglucose and the sample may be introduced to a sensor strip. The methodmay include transferring at least one electron from or to the analyte inthe sample to form the measurable species, which may include at leastone mediator.

The input signal may include at least two excitations separated by arelaxation, where the at least two excitations have durations from 0.1to 5 seconds and the duration of the relaxation is at least 0.1 secondor at least 0.5 second. Each excitation and/or relaxation duration maybe the same or different. The duration for one or more of therelaxations may be from 0.1 to 3 seconds. The input signal may includeat least three excitations and at least two relaxations. The inputsignal may include at least 2 duty cycles applied within 5 seconds.

The incubation period may be from 0.1 to 8 seconds, from 0.1 to 6seconds, or from 0.5 to 4.75 seconds, for example. The incubation periodand the application of the input signal may be complete in at most 12,at most 6, or at most 4 seconds. The transient decay may have a decayconstant from −0.52 to −1, or from −0.001 to −0.48. The transient decaymay have a decay constant of at most −0.45 or at most −0.35. The outputsignal from which the analyte concentration is determined may include acurrent value recorded within 2 seconds of applying the input signal tothe sample. The analyte concentration of the sample may be determinedwithin at most 6, 3, or 1.5 seconds of applying the input signal.

The sample may reside in a reservoir defined by a sensor strip base andthe bottom surface of a lid, the base being 20 to 200 micrometers fromthe bottom surface of the lid. The volume of sample within the reservoirmay be from 0.25 to 10 microliters for from 0.25 to 1.5 microliter. Thereservoir may include at least one reagent layer having an averageinitial thickness of at most 20 micrometers, less than 14 micrometers,or at most 5 micrometers. The reservoir may include at least one reagentlayer having an average initial thickness of at most 2 micrometers whenthe input signal includes at least two excitations, at least one of theexcitations having a duration of at most 0.5 seconds. The reservoir mayinclude at least one reagent layer comprising a distinct diffusionbarrier layer.

The reservoir height from the sensor strip base to the bottom of the lidmay be at most 250 micrometers, the volume of the sample within thereservoir may be at most 5 microliters, the reservoir may include atleast one reagent layer having an average initial thickness of at most20 micrometers, and the incubation period may be at most 12 seconds. Thereservoir height from the sensor strip base to the bottom of the lid maybe at most 150 micrometers, the volume of the sample within thereservoir may be at most 3.5 microliters, the reservoir may include atleast one reagent layer having an average initial thickness of less than14 micrometers, and the incubation period may be at most 6 seconds. Thereservoir height from the sensor strip base to the bottom of the lid maybe at most 100 micrometers, the volume of the sample within thereservoir may be at most 3 microliters, the reservoir may include atleast one reagent layer having an average initial thickness of at most 2micrometers, and the incubation period may be at most 2 seconds.

In another aspect, a method for determining an analyte concentration ina sample includes applying an input signal to the sample after anincubation period of at most 12 seconds, generating an output signalhaving a transient decay in response to a redox reaction of a measurablespecies; and determining the analyte concentration from the outputsignal.

In another aspect, a biosensor for determining an analyte concentrationin a sample includes a measurement device having a processor connectedto a sensor interface; a sensor strip having a sample interface on abase, the sensor interface in electrical communication with the sampleinterface, where the sample interface is adjacent to a reservoir formedby the base; where the processor instructs a charger to apply an inputsignal to the reservoir after an incubation period of at most 12seconds; and where the processor determines the analyte concentration inthe sample from an output signal having a transient decay in response toa redox reaction of the analyte in the sample.

The reservoir may include at least one working electrode in electricalcommunication with the charger, a reagent layer on the working electrodehaving a combination DBL/reagent layer with an average initial thicknessfrom about 1 micrometer to about 20 micrometers. The combinationDBL/reagent layer may have an average initial thickness of at most 1micrometer.

In another aspect, a method for determining an analyte concentration ina sample includes applying an input signal to the sample after anincubation period of at most 12 seconds; generating a variantconcentration distribution of a measurable species in a samplereservoir; generating an output signal in response to a redox reactionof a measurable species; and determining the analyte concentration fromthe output signal.

In another aspect, a method for determining an analyte concentration ina sample includes introducing the sample to a sensor strip; applying aninput signal to the sample after an incubation period of at most 8seconds; generating an output signal having a transient decay inresponse to a redox reaction of a measurable species; and determiningthe analyte concentration from the transient decay of the output signal.The transient decay may be a decreasing current decay obtained within0.5 to 5 seconds or in about 0.5 to about 3 seconds of applying theinput signal to the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention can be better understood with reference to the followingdrawings and description. The components in the figures are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1A is a perspective representation of an assembled sensor strip.

FIG. 1B is a top-view diagram of a sensor strip, with the lid removed.

FIG. 2A is an end view diagram of the sensor strip of FIG. 1B.

FIG. 2B depicts a schematic representation of a biosensor system thatdetermines an analyte concentration in a sample.

FIG. 3 represents a flowchart of an electrochemical method fordetermining the presence and/or concentration of an analyte in a sample.

FIG. 4A represents a sample reservoir bounded by a lower electrodesurface and an upper lid.

FIG. 4B represents concentration profiles formed from the sensor systemwhen incubation times t₁ through t₅ pass before application of an inputsignal.

FIG. 4C represents the relation between measurable speciesconcentrations in the reservoir and rates of current decay.

FIG. 5 depicts decay rates obtained from working electrodes aftervarying incubation periods for whole blood samples containing 50, 100,200, or 400 mg/dL of glucose.

FIGS. 6A-6C plot the current profiles obtained from three sensor stripseach having a different average initial thickness of the reaction layerat multiple initial incubation periods.

FIGS. 7A-7B plot the natural logs of current vs. time for whole bloodsamples including 100 or 300 mg/dL of glucose at 40% hematocrit obtainedafter a 6 second initial incubation period.

FIGS. 8A-8C are current decay profiles from a 0.25 second incubationperiod followed by a gated input signal having excitation times of 0.5second and relaxation times of 0.25 second.

FIG. 8D is a calibration plot obtained by plotting the endpoint currents(p1, p2, p3) of the first three excitations obtained from thin reagentlayer sensor strips as depicted in FIGS. 8A-8C.

FIG. 8E is a calibration plot obtained by plotting the endpoint currents(p4, p5, p6) of excitations 4, 5, and 6 obtained from sensor stripshaving intermediate thickness reagent layers as depicted in FIGS. 8A-8C.

DETAILED DESCRIPTION

A biosensor system uses an electrochemical process lacking a Cottrelldecay constant to determine an analyte concentration of a biologicalsample. The biosensor system generates an output signal from thebiological sample having a transient decay, where the output signal isnot inversely related to the square root of the time. The transientdecay output from the biosensor system has a decay constant greater orless than −0.5 and the system does not rely on an estimation of a steadystate current value to determine the analyte concentration. Preferably,transient decays from which analyte concentrations are determinedcontinually decrease.

Cottrell decay is diffusion dependent and may not exist unless theanalyte has completely converted to a measurable species and asubstantially constant concentration distribution of this measurablespecies occupies the sample reservoir before current measurement.Relatively long incubation times and large sample volumes are requiredto obtain Cottrell decay. Without these conditions, the output currentwill not be inversely related to the square root of time and thusbiosensors will not exhibit the −0.5 decay constant required forCottrell decay. Biosensors designed to operate with Cottrell decay willprovide inaccurate analyses if the output current is not inverselyrelated to the square root of time or if a decay constant other than−0.5 is present in the output signal.

The present biosensor system operates using transient decays, wheredecay constants smaller or larger than −0.5 are observed. The transientand thus non-Cottrell decay constants may result from a relatively shortincubation period. Transient decay constants also may result fromrelatively small sample reservoir volumes, relatively small distancesbetween electrode surfaces and the lid of the sensor strip, and/orrelatively short excitations in relation to the average initialthickness of the reagent layer.

To generate an output current with a transient decay or transient decayconstants greater or less than −0.5, the biosensor system may useincubation periods of 12 seconds or less, reservoir volumes of 5 μL orless, reservoir heights of 200 μm or less, and/or an average initialthickness for the reagent layer of 20 μm or less. Preferable incubationperiods for use with reservoir volumes of 3.5 μL or less, reservoirheights of 150 μm or less, and/or an average initial thickness for thereagent layer of 10 μm or less are at most 8 seconds, at most 6 seconds,or at most 4 seconds. At present, especially preferred incubationperiods for use with sample strip sample volumes of 3.0 μL or less,sample strip cap-gap heights of 100 μm or less, and/or an averageinitial thickness for the reagent layer of 2 μm or less are at most 2seconds or at most 1 second. Other incubation periods, reservoirvolumes, reservoir heights, and reagent layer thicknesses may be used.

FIGS. 1A and 1B depict a sensor strip 100, which may be used with thebiosensor system. FIG. 1A is a perspective view of an assembled sensorstrip 100 including a sensor base 110, at least partially covered by alid 120 that includes a vent 130, a sample coverage area 140, and aninput end opening 150. A partially-enclosed sample reservoir 160 (thecapillary gap or cap-gap) is formed between the base 110 and the lid120. Other sensor strip designs also may be used, such as thosedescribed in U.S. Pat. Nos. 5,120,420 and U.S. Pat. Nos. 5,798,031.While a particular configuration is shown in FIGS. 1A-1B, the sensorstrip 100 may have other configurations, including those with additionalcomponents.

The height of the reservoir 160 between the sensor base 110 and the lid120 may be from 20 to 250 micrometers (μm), more preferably from 50 to150 μm. The volume of the reservoir 160 may be from 0.25 to 10 μL,preferably from 0.8 to 4 μL, and more preferably from 0.5 to 1.5 μL.Other heights and volumes may be used.

A liquid sample for analysis may be transferred into the reservoir 160by introducing the liquid to the opening 150. The liquid fills thereservoir 160 while expelling the previously contained air through thevent 130. The reservoir 160 may contain a composition (not shown) thatassists in retaining the liquid sample in the reservoir. Examples ofsuch compositions include: water-swellable polymers, such ascarboxymethyl cellulose and polyethylene glycol; and porous polymermatrices, such as dextran and polyacrylamide.

FIG. 1B depicts a top-view of the sensor strip 100, with the lid 120removed. Conductors 170 and 180 may run under a dielectric layer 190from the opening 150 to a working electrode 175 and a counter electrode185, respectively. The sensor strip 100 may include more than oneworking electrode. The working and counter electrodes 175, 185 may be insubstantially the same plane. The electrodes may be in anotherorientation. The dielectric layer 190 may partially cover the electrodes175, 185 and may be made from any suitable dielectric material, such asan insulating polymer. While a particular electrode configuration isshown, the electrodes may have other configurations, including thosewith additional components.

The counter electrode 185 may support the electrochemical activity atthe working electrode 175 of the sensor strip 100. The potential tosupport the electrochemical activity at the working electrode 175 may beprovided to the sensor system by forming the counter electrode 185 froman inert material, such as carbon, and including a soluble redoxspecies, such as ferricyanide, within the reservoir 160. The potentialat the counter electrode 185 may be a reference potential achieved byforming the counter electrode 185 from a redox pair, such as Ag/AgCl, toprovide a combined reference-counter electrode. A redox pair includestwo conjugate species of a chemical substance having different oxidationnumbers. Reduction of the species having the higher oxidation numberproduces the species having the lower oxidation number. Alternatively,oxidation of the species having the lower oxidation number produces thespecies having the higher oxidation number. The sensor strip 100 may beprovided with a third conductor and electrode to provide a referencepotential to the sensor system.

The working and counter electrodes 175, 185 may be separated by greaterthan 200 μm or 250 μm. The working and counter electrodes 175, 185 maybe separated by less than 200 μm. The working and counter electrodes175, 185 may be separated by other distances.

FIG. 2A depicts an end view of the sensor strip 100 depicted in FIG. 1B,showing the layer structure of the working electrode 175 and the counterelectrode 185 residing within the reservoir 160. The conductors 170 and180 may lie on the base 110. Other materials may reside between theconductors 170, 180 and the base 110, thus the conductors may or may notbe in physical contact with the base. A portion of the conductors maypenetrate a portion of the base. Surface conductor layers 270 and 280optionally may be deposited on the conductors 170 and 180, respectively.Other materials may reside between the surface conductor layers 270, 280and the conductors 170, 180, thus the surface conductors may or may notbe in physical contact with the conductors. A portion of the surfaceconductors may penetrate a portion of the conductors. The surfaceconductor layers 270, 280 may be made from the same or from differentmaterials.

The material or materials forming the conductors 170, 180 and thesurface conductor layers 270, 280 may include any electrical conductor.The conductors 170, 180 preferably include a thin layer of a metal pasteor metal, such as gold, silver, platinum, palladium, copper, ortungsten. The surface conductor layers 270, 280 preferably includecarbon, gold, platinum, palladium, or combinations thereof. Preferableelectrical conductors are non-ionizing, such that the material does notundergo a net oxidation or a net reduction during analysis of thesample. Thus, if a surface conductor layer is not on a conductor, theconductor is preferably made from a non-ionizing material, such ascarbon, gold, platinum, palladium, or combinations thereof.

The surface conductor material may be deposited on the conductors 170,180 by any conventional means compatible with the operation of thesensor strip, including foil deposition, chemical vapor deposition,slurry deposition, and the like. In the case of slurry deposition, theconductor material may be applied as an ink to the conductors 170, 180,as described in U.S. Pat. No. 5,798,031.

The reagent layers 275 and 285 may be deposited on the conductors 170and 180, respectively. The layers are formed from at least one reagentcomposition that may include a binder. The binder is preferably apolymeric material that is at least partially water-soluble. The bindermay form a gel or gel-like material when hydrated. The binder may form agel or gel-like material in combination with the reagents when hydrated.The gel or gel-like material may inhibit and/or filter red blood cellsfrom reaching the surface conductor 270 and/or the conductor 170.

Suitable partially water-soluble polymeric materials for use as thebinder may include poly(ethylene oxide) (PEO), carboxymethyl cellulose(CMC), polyvinyl alcohol (PVA), hydroxyethyl cellulose (HEC),hydroxypropyl cellulose (HPC), methyl cellulose, ethyl cellulose, ethylhydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinylpyrrolidone (PVP), polyamino acids, such as polylysine, polystyrenesulfonate, gelatin, acrylic acid, methacrylic acid, starch, maleicanhydride salts thereof, derivatives thereof, and combinations thereof.Among the above binder materials, PEO, PVA, CMC, and HEC are preferred,with CMC being more preferred at present.

In addition to the binder, the reagent layers 275 and 285 may includethe same or different reagents. When including the same reagents, thereagent layers 275 and 285 may be the same layer. In one aspect, thereagents present in the first layer 275 may be selected for use with theworking electrode 175, while the reagents present in the second layer285 may be selected for use with the counter electrode 185. For example,the reagents in the layer 285 may facilitate the flow of electronsbetween the sample and the conductor 180. Similarly, the reagents in thelayer 275 may facilitate the reaction of the analyte.

The reagent layer 275 may include an enzyme system specific to theanalyte that may enhance the specificity of the sensor system to theanalyte, especially in complex biological samples. The enzyme system mayinclude one or more enzyme, cofactor, and/or other moiety thatparticipates in the redox reaction of the analyte. For example, analcohol oxidase can be used to provide a sensor strip that is sensitiveto the presence of alcohol in a sample. Such a system may be useful inmeasuring blood alcohol concentrations. In another example, glucosedehydrogenase or glucose oxidase may be used to provide a sensor stripthat is sensitive to the presence of glucose in a sample. This systemmay be useful in measuring blood glucose concentrations, for example inpatients known or suspected to have diabetes.

Enzymes for use in the enzyme system include alcohol dehydrogenase,lactate dehydrogenase, β-hydroxybutyrate dehydrogenase,glucose-6-phosphate dehydrogenase, glucose dehydrogenase, formaldehydedehydrogenase, malate dehydrogenase, and 3-hydroxysteroid dehydrogenase.Preferable enzyme systems may be oxygen independent, thus notsubstantially oxidized by oxygen.

One such oxygen independent enzyme family for use in a glucose sensorstrip is glucose dehydrogenase (GDH). Using different co-enzymes orco-factors, GDH may be mediated in a different manner by differentmediators. Depending on the association with GDH, a co-factor, such asflavin adenine dinucleotide (FAD), can be tightly held by the hostenzyme, such as in the case of FAD-GDH; or a co-factor, such asPyrroloquinoline quinone (PQQ), may be covalently linked to the hostenzyme, such as with PQQ-GDH. The co-factor in each of these enzymesystems may be held by the host enzyme, or the co-enzyme and theapo-enzyme may be re-constituted before the enzyme system is added tothe reagent composition. The co-enzyme also may be independently addedto the host enzyme in the reagent composition to assist in the catalyticfunction of the host enzyme, such as in the cases of nicotinamideadenine dinucleotide NAD/NADH⁻ or nicotinamide adenine dinucleotidephosphate NADP/NADPH⁺.

The reagent layer 275 also may include a mediator to more effectivelycommunicate the results of the analyte redox reaction to the surfaceconductor 270 and/or the conductor 170. Mediators may be separated intotwo groups based on their electrochemical activity. One electrontransfer mediators are capable of taking on one additional electronduring electrochemical reactions. Examples of one electron transfermediators include compounds, such as 1,1′-dimethyl ferrocene,ferrocyanide and ferricyanide, and ruthenium(III) hexaamine. Twoelectron transfer mediators are capable of taking on two additionalelectrons.

Two electron mediators include the organic quinones and hydroquinones,such as phenanthroline quinone; phenothiazine and phenoxazinederivatives; 3-(phenylamino)-3H-phenoxazines; phenothiazines; and7-hydroxy-9,9-dimethyl-9H-acridin-2-one and its derivatives. Examples ofadditional two electron mediators include the electroactive organicmolecules described in U.S. Pat. Nos. 5,393,615; 5,498,542; and U.S.Pat. Nos. 5,520,786, which are incorporated herein by reference. Otherelectroactive organic molecules include organic molecules lacking ametal that are capable of undergoing a redox reaction. Electroactiveorganic molecules can behave as redox species and/or as mediators.Examples of electro-active organic molecules include coenzymepyrroloquinoline quinone (PQQ), benzoquinones and naphthoquinones,N-oxides, nitroso compounds, hydroxylamines, oxines, flavins,phenazines, phenothiazines, indophenols, and indamines.

Preferred two electron transfer mediators include3-phenylimino-3H-phenothiazines (PIPT) and 3-phenylimino-3H-phenoxazines(PIPO). More preferred two electron mediators include the carboxylicacid or salt, such as ammonium salts, of phenothiazine derivatives. Atpresent, especially preferred two electron mediators include(E)-2-(3H-phenothiazine-3-ylideneamino)benzene-1,4-disulfonic acid,(E)-5-(3H-phenothiazine-3-ylideneamino)isophthalic acid, ammonium(E)-3-(3H-phenothiazine-3-ylideneamino)-5-carboxybenzoate, andcombinations thereof. Preferred two electron mediators may have a redoxpotential that is at least 100 mV lower, more preferably at least 150 mVlower, than ferricyanide.

The reagent layers 275, 285 may be deposited by any convenient means,such as printing, liquid deposition, or ink jet deposition. In oneaspect, the layers are deposited by printing. With other factors beingequal, the angle of the printing blade may inversely affect the initialthickness of the reagent layer. For example, when the blade is moved atan approximately 82° angle to the base 110, the layer may have aninitial thickness of approximately 10 μm. Similarly, when a blade angleof approximately 62° to the base 110 is used, a thicker 30 μm layer maybe produced. Thus, lower blade angles may provide thicker reagentlayers. In addition to blade angle, other factors, such as the viscosityof the reagent composition as well as the screen-size and emulsioncombination, may affect the resulting thickness of the reagent layers275, 285.

When thinner reagent layers are preferred, deposition methods other thanprinting, such as micro-pipetting, ink jetting, or pin-deposition, maybe used. These deposition methods generally give the dry reagent layersat micrometer or sub-micrometer thickness, such as 1-2 μm. For example,pin-deposition methods may provide an average initial thickness of about1 μm for the reagent layer. The thickness of the reagent layer resultingfrom pin-deposition, for example, may be controlled by the amount ofpolymer included in the reagent composition, with higher polymer contentproviding thicker reagent layers. Thinner reagent layers may requireshorter excitation durations than thicker reagent layers to maintain thedesired measurement performance and/or substantially measure analytewithin the diffusion barrier layer (DBL).

The working electrode 175 may include a DBL that is integral to areagent layer 275 or that is a distinct layer 290, such as depicted inFIG. 2A. Thus, the DBL may be formed as a combination reagent/DBL on theconductor, as a distinct layer on the conductor, or as a distinct layeron the reagent layer. When the working electrode 175 includes thedistinct DBL 290, the reagent layer 275 may or may not reside on the DBL290. Instead, the reagent layer 275 may reside on any portion of thesensor strip 100 that allows the reagent to solubilize in the sample.For example, the reagent layer 175 may reside on the base 110 or on thelid 120.

The DBL provides a porous space having an internal volume where ameasurable species may reside and also may filter red blood cells fromthe conductor surface. The pores of the DBL may be selected so that themeasurable species may diffuse into the DBL, while physically largersample constituents, such as red blood cells, are substantiallyexcluded. Although conventional sensor strips have used variousmaterials to filter red blood cells from the surface of the workingelectrode, a DBL provides an internal porous space to contain andisolate a portion of the measurable species from the sample.

When the reagent layer 275 includes a water-soluble binder, any portionof the binder that does not solubilize into the sample prior to theapplication of an excitation may function as an integral DBL. Theaverage initial thickness of a combination DBL/reagent layer ispreferably less than 20 or 10 μm and more preferably less than 5 μm. Thedesired average initial thickness of a combination DBL/reagent layer maybe selected for a specific excitation length on the basis of when thediffusion rate of the measurable species from the DBL to a conductorsurface, such as the surface of the conductor 170 or the surface of thesurface conductor 270 from FIG. 2A, becomes relatively constant. Thecombination DBL/reagent layer may have an average initial thickness of 2μm, 1 μm, or less when combined with an excitation duration of 0.25seconds or less.

The distinct DBL 290 may include any material that provides the desiredpore space, while being partially or slowly soluble in the sample. Thedistinct DBL 290 may include a reagent binder material lacking reagents.The distinct DBL 290 may have an average initial thickness from 1 to 15μm, and more preferably from 2 to 5 μm.

FIG. 2B depicts a schematic representation of a biosensor system 200that determines an analyte concentration in a sample, such as abiological fluid. The biosensor system 200 includes a measurement device202 that performs an analysis method and a sensor strip 204. The sensorstrip 204 may be an electrochemical sensor strip as depicted in FIGS.1A, 1B, and 2A, for example. The measurement device 202 may beimplemented as a bench-top device, a portable or hand-held device, orthe like. The measurement device 202 and the sensor strip 204 mayimplement an electrochemical analysis, an optical analysis, acombination thereof, or the like. The biosensor system 200 may determineanalyte concentrations, including those of alcohol, glucose, uric acid,lactate, cholesterol, bilirubin, and the like in biological samples.While a particular configuration is shown, the biosensor system 200 mayhave other configurations, including those with additional components.

The sensor strip 204 has a base 206 that forms a sample reservoir 208and a channel 210 with an opening 212. Referring to FIG. 1A, the channel210 may be integral to the reservoir 208. The reservoir 208 and thechannel 210 may be covered by a lid with a vent. The reservoir 208defines a partially-enclosed volume (the cap-gap). The reservoir 208 maycontain a composition that assists in retaining a liquid sample, such aswater-swellable polymers or porous polymer matrices. Reagents may bedeposited in the reservoir 208 and/or channel 210. The reagentcomposition may include one or more enzymes, binders, mediators, and thelike. The reagents may include a chemical indicator for an opticalsystem. The sensor strip 204 may have other configurations.

The sensor strip 204 also may have a sample interface 214. In anelectrochemical system, the sample interface 214 has conductorsconnected to at least two electrodes, such as a working electrode and acounter electrode. The electrodes may be disposed on a surface of thebase 206 that forms the reservoir 208. The sample interface 214 may haveother electrodes and/or conductors.

The measurement device 202 includes electrical circuitry 216 connectedto a sensor interface 218 and a display 220. The electrical circuitry216 may include a processor 222 connected to a signal generator 224, anoptional temperature sensor 226, and a storage medium 228. Theelectrical circuitry 216 may have other configurations including thosewith additional components.

The signal generator 224 provides an electrical input signal to thesensor interface 218 in response to the processor 222. In opticalsystems, the electrical input signal may be used to operate or controlthe detector and light source in the sensor interface 218. Inelectrochemical systems, the electrical input signal may be transmittedby the sensor interface 218 to the sample interface 214 to apply theelectrical input signal to the reservoir 208 and thus, to the sample.

The electrical input signal may be a potential or current and may beconstant, variable, or a combination thereof, such as when an AC signalis applied with a DC signal offset. The electrical input signal may beapplied as a single pulse or in multiple pulses, sequences, or cycles.The signal generator 224 also may record an output signal from thesensor interface 218 as a generator-recorder.

The storage medium 228 may be a magnetic, optical, or semiconductormemory, another computer readable storage device, or the like. Thestorage medium 228 may be a fixed memory device or a removable memorydevice such as a memory card.

The processor 222 may implement analyte analysis and data treatmentusing computer readable software code and data stored in the storagemedium 228. The processor 222 may start the analyte analysis in responseto the presence of the sensor strip 204 at the sensor interface 218, theapplication of a sample to the sensor strip 204, in response to userinput, or the like. The processor 222 may direct the signal generator224 to provide the electrical input signal to the sensor interface 218.The processor 222 may receive the sample temperature from thetemperature sensor 226, if so equipped.

The processor 222 receives the output signal from the sensor interface218. The output signal is generated in response to the redox reaction ofthe analyte in the sample. The output signal may be generated using anoptical system, an electrochemical system, or the like. The processor222 may determine the concentration of the analyte in the sample fromone or more output signals using a correlation equation. The results ofthe analyte analysis are output to the display 220 and may be stored inthe storage medium 228.

The correlation equations relating analyte concentrations and outputsignals may be represented graphically, mathematically, a combinationthereof, or the like. The correlation equations may be represented by aprogram number assignment (PNA) table, another look-up table, or thelike that is stored in the storage medium 228. Instructions regardingimplementation of the analysis may be provided by the computer readablesoftware code stored in the storage medium 228. The code may be objectcode or any other code describing or controlling the functionalitydescribed herein. The data from the analyte analysis may be subjected toone or more data treatments, including the determination of decay rates,K constants, slopes, intercepts, and/or sample temperature in theprocessor 222.

In electrochemical systems, the sensor interface 218 is in electrical oroptical communication with the sample interface 214. Electricalcommunication includes the transfer of input and/or output signalsbetween contacts in the sensor interface 218 and conductors in thesample interface 214. Electrical communication may be implementedwirelessly or through physical contact, for example. The sensorinterface 218 transmits the electrical input signal from the signalgenerator 224 through the contacts to the connectors in the sampleinterface 214. The sensor interface 218 also transmits the output signalfrom the sample through the contacts to the processor 222 and/or thesignal generator 224.

Optical communication includes the transfer of light between an opticalportal in the sample interface 202 and a detector in the sensorinterface 208. Optical communication also includes the transfer of lightbetween an optical portal in the sample interface 202 and a light sourcein the sensor interface 208.

The display 220 may be analog or digital. The display 220 may be a LCD,LED, or vacuum fluorescent display adapted to displaying a numericalreading.

In use, a liquid sample for analysis is transferred into the reservoir208 by introducing the liquid to the opening 212. The liquid sampleflows through the channel 210 and into the reservoir 208, whileexpelling the previously contained air. The liquid sample chemicallyreacts with the reagents deposited in the channel 210 and/or thereservoir 208. The processor 222 directs the signal generator 224 toprovide an input signal to the sensor interface 218. In an opticalsystem, the sensor interface 218 operates the detector and light sourcein response to the input signal. In an electrochemical system, thesensor interface 218 provides the input signal to the sample through thesample interface 214. The processor 222 receives the output signalgenerated in response to the redox reaction of the analyte in thesample. The processor 222 determines the analyte concentration of thesample using one or more correlation equations. The determined analyteconcentration may be displayed and/or stored for future reference.

FIG. 3 represents a flowchart of an electrochemical analysis 300 fordetermining the presence and optionally the concentration of an analyte322 in a sample 312. In 310, the sample 312 is introduced to a sensorstrip 314, such as the sensor strip depicted in FIGS. 1A-1B and 2A. Thereagent layers, such as 275 and/or 285 depicted in FIG. 2A, begin tosolubilize into the sample 312, thus allowing reaction.

In 315, an initial incubation period 317 allows the reagents to reactwith the sample 312 before an input signal is applied. Preferably, theincubation period 317 may be from 0.1 to 10 seconds, more preferablyfrom 0.1 to 8 seconds or from 0.5 to 4 seconds. At present, from 0.1 to1 second is more preferred for the incubation period 317. Otherincubation periods may be used.

During the incubation period 317, a portion of the analyte 322 presentin the sample 312 is chemically or biochemically oxidized or reduced in320 by way of a redox reaction to form a measurable species 332. Themeasurable species 332 may be the oxidized or reduced analyte 322 or amediator. Upon oxidation or reduction, electrons may be transferred toor from the analyte 322 and to or from measurable species 332 in 330.For example, a mediator may be reduced to form the measurable species332 through oxidation of the analyte 322. Preferably, the measurablespecies 332 formed during the incubation period 317 is notelectrochemically excited during the incubation period 317.

In 340, the measurable species 332 is electrochemically excited(oxidized or reduced). In this manner, electrons are selectivelytransferred between the analyte 322 and the working electrode of thesensor strip 314. The excitation 340 may be from 0.1 to 5 seconds orfrom 0.1 to 1 second in duration. The excitation 340 may be repeated.

In 350, the current produced during the excitation 340 may be recordedas a function of time. If multiple excitations 340 are applied to thesensor strip 314, one or more of the currents resulting from theexcitations 340 may be recorded in 350. The currents may be recorded bya measurement device.

In 360, the sample undergoes relaxation. Preferably, current is notrecorded during the relaxation 360. The relaxation 360 may follow eachof the excitations 340 when multiple excitations are applied. During therelaxation 360, the current present during the excitation 340 issubstantially reduced by at least one-half, preferably by an order ofmagnitude, and more preferably to zero. Preferably, a zero current stateis provided by an open circuit or other method known to those ofordinary skill in the art to provide a substantially zero current flow.The measurement device may open the circuit through the sensor strip 314to provide the open circuit. If a zero current state is provided, therelaxation 360 may be considered an intermittent incubation period.

The relaxation 360 may be at least 0.1 or at least 0.5 seconds induration. The relaxation 360 may be from 0.1 to 3 seconds, from 0.2 to 2seconds, or from 0.5 to 1 second in duration. Other relaxation durationsmay be used.

In 370, one or more of the recorded current and time values from 350 maybe analyzed to determine the presence and/or concentration of theanalyte 322 in the sample 312. Preferably, the analyte concentration isdetermined from a current measurement taken within 2 seconds or 1 secondof the start of the initially applied excitation. More preferably,multiple short excitations are combined with a current measurement takenwithin 2 seconds, 1 second, or less from the start of the initiallyapplied input signal to determine the analyte concentration of thesample. The recorded current and time values may be correlated to theconcentration of the analyte 322 in the sample 312 using one or morecorrelation equations.

The excitation 340 and the relaxation 360 constitute a single dutycycle. Preferably, the input signal applied to the sensor strip 314includes at least 2, 4, or 6 duty cycles applied within an independentlyselected 3, 5, 7, or 9 second time period. Thus, from the initialapplication of the input signal, the total time required for theexcitation 340 and the relaxation 360 portions of the electrochemicalanalysis 300 may be at most 3, at most 5, at most 7, or at most 9seconds. The duty cycles may be applied during a 1 to 3 second timeperiod. From 2 to 6 duty cycles may be applied within 8 seconds or less.From 2 to 4 duty cycles may be applied within 3 to 6 seconds. Other timeperiods may be used.

For continuous monitoring, as may be used with implanted or partiallyimplanted sensors, the duty cycles may be continuously repeated. Theenergy required to operate the system may be reduced and the servicelife of the system may be extended in relation to methods lackingrelaxations. Furthermore, the application of multiple duty cycles may beseparated by longer time periods, such as 5 minutes or more.

Amperometric sensor systems apply a potential (voltage) to theelectrodes to excite the measurable species while the current (amperage)is monitored. Conventional amperometric sensor systems may maintain theexcitation potential while continuously measuring the current for from 5to 10 seconds, for example. In contrast to conventional methods, theinput signals used in the electrochemical analysis 300 may replacecontinuous, long-duration excitations with multiple excitations andrelaxations of relatively short duration. A more detailed description ofmultiple excitation and relaxation or “gated” pulse sequences applied asinput signals may be found in WO 2007/013915, filed Jul. 19, 2006,entitled “Gated Amperometry.”

When the short initial incubation times and/or gated input signals ofthe present invention are used, transient or non-Cottrell current decaysmay result. Not relying on a −0.5 Cottrell decay constant to determinethe concentration of the analyte 322 in the sample 312 allows forcompletion of the electrochemical analysis 300 using transient decayswithin 8 seconds or less, within 4 seconds or less, or more preferably,within 3 seconds or less. The electrochemical analysis 300 may becompleted in 2 seconds or less. The electrochemical analysis 300 may becompleted in from about 0.5 to about 3 seconds. The electrochemicalanalysis 300 using transient decays may be complete using other timeperiods.

FIG. 4A represents a sample reservoir 400 bounded by a lower electrodesurface 402 and an upper lid 403. A virtual upper boundary 405 of thereagent layer also is represented. Thus, the area between the electrodesurface 402 and the virtual upper boundary 405 represents the samplecontained by the reagent layer. Similarly, the area between the virtualupper boundary 405 and the upper lid 403 represents the sample above thereagent layer. The x-axis represents distance from the electrodesurface, while the y-axis represents the sample concentration ofmeasurable species generated from the redox reaction of the analyte. Thefigure omits the effect of analyte partitioning between a DBL and theliquid sample within the remaining portion of the reservoir 400.

Concentration profile 410 represents what would be observed immediatelyafter introducing the sample to a strip, while concentration profile 420represents what would be observed after a relatively long incubationperiod. The concentration profile 410 represents a transient condition,while the concentration profile 420 represents a Cottrell condition.Multiple transient states may exist between the transient concentrationprofile 410 and the Cottrell concentration profile 420.

FIG. 4B represents the formation of different concentration profileswhen incubation times t₁ through t₅ pass before the input signal isapplied to the electrodes. The concentration profile at t₅, representinga 15 to 30 second incubation period, depicts a substantially constantconcentration distribution of measurable species throughout the sample,which would provide a Cottrell decay having a decay constant of −0.5.Thus, the area under the t₅ line and the related measurable speciesconcentration does not substantially change until a relatively largedistance away from the electrode surface 402.

In contrast to the t₅ line, the t₄ line has an incubation period of 1 to12 seconds and a variant concentration distribution of measurablespecies in the sample. The t₄ line has slower transient decay constantsfrom −0.30 (1 second) to −0.48 (12 seconds). Thus, the area under the t₄line and the underlying measurable species concentration undergoes asubstantial change from the electrode surface 402 to the upper lid 403of the reservoir 400—thus being variant.

As the incubation period is further reduced to 0.4 to 1 second in t₃ orto 0.1 to 0.3 second in t₂, the transient decay constants may range from−0.25 to −0.3 for t₃ and from −0.15 to −0.25 for t₂, respectively. Thet₁ decay, representing a 0.01 to 0.1 second incubation period may have atransient decay constant of −0.15 or less. As the incubation period isreduced from t₄ to t₁, the area under the lines and the relatedmeasurable species concentration between the electrode surface 402 andthe upper lid 403 of the reservoir 400 becomes increasingly variant.

By having a lower concentration of the measurable species at theelectrode surface 402 than in the remaining portion of the reservoir400, such as represented by the t₁ through t₄ variant concentrationdistribution profiles of FIG. 4B, the rate of current decay may beslower than the −0.5 decay constant required for Cottrell decay. Thisslower decay may be attributable to the large concentration ofmeasurable species farther from the electrode surface 402 reaching theelectrode surface more rapidly than if the measurable species wasdistributed evenly throughout the sample reservoir 400. Similarly,faster decay rates may be obtained when a higher concentration of themeasurable species is present at the electrode surface 402 than in theremaining portion of the sample reservoir 400.

FIG. 4C represents the relation between measurable speciesconcentrations in the reservoir 400 and current decay constants.Measurable species concentration profiles 430 and 440 have slower andfaster decay rates, respectively, than 420, which corresponds to the−0.5 Cottrell decay constant. For concentration profile 430 having adecay constant less than the −0.5 Cottrell decay constant, such as −0.3,the rate of current decay will be slower than that observed for aCottrell system. Similarly, for concentration profile 440 having a decayconstant greater than the −0.5 Cottrell decay constant, such as −0.7,the rate of current decay will be faster than that observed for aCottrell system. Thus, in comparison to the −0.5 Cottrell decay constantrepresented by 420, transient decay constants 430, 440 reflect variantconcentration distributions of the measurable species in the reservoir400.

When long incubation periods are used to generate Cottrell decay, theamount of measurable species produced during the measurement excitationis small compared to the amount of measurable species produced duringthe prior incubation period. Thus, unlike the concentration profile 420representing complete redox conversion of the analyte to a measurablespecies before application of the input signal, concentration profiles430, 440 represent incomplete conversion. Furthermore, any change indiffusion rate of the measurable species to the electrode fromconvection or other pathways also is small in relation to the amount ofmeasurable species generated during the incubation period. Thus, longincubation periods substantially negate effects that would alter the−0.5 Cottrell decay constant.

In contrast, when short incubation periods, such as 12 seconds, 10seconds, and shorter are used, the amount of measurable species producedduring the measurement excitation and any change in diffusion rates fromprocesses other than diffusion may provide an actual decay rate that isslower than the −0.5 Cottrell value. This decay process can be describedby the following normalized current equation, Equation (2):f(t)=t^(−a+b+c)   (2),where a is the portion of the decay constant from measurable speciesformed during the incubation period, b is the portion of the decayconstant from measurable species formed during the measurementexcitation, and c is the portion of the decay constant arising fromvariations in the concentration distribution of the measurable speciesin the sample reservoir. Negative values of b and c result in anincrease in measured measurable species concentration, while positivevalues of b and c result in a decrease in measured measurable speciesconcentration. Thus, if either a or b are non-zero, a deviation from thea decay value will result. As a Cottrell decay is provided by a −0.5value for a, a significant contribution from b or c provides a transientdecay constant. Under Equation (2), term a controls the decay constantobtained from the concentration profile 420, while term b wouldsignificantly contribute to the decay constant obtained from theconcentration profiles 430 and 440, where the input signal is appliedbefore redox conversion of the analyte is complete.

Equation (2) establishes that the decay constant of a system can varyover time in response to which of these underlying factors affect thecurrent decay at the time of measurement. For example, longer incubationperiods increase a while reducing b because the more analyte convertedto the measurable species during the incubation period, the less analyteremains in the sample for conversion to the measurable species duringthe excitation.

The redox conversion of analyte to measurable species occurs in hydratedreagent layers. Because thicker reagent layers require longer tohydrate, thicker reagent layers will provide an increase in the b termin relation to the a term if the input signal is applied before thereagent layer is hydrated. Cottrell decay is not observed before thereagent layer is hydrated due to the contribution to the decay constantof measurable species formed during the measurement excitation, the bterm of Equation (2). This was recognized in column 4, lines 58-59 ofthe '069 patent, which discloses that incomplete wetting of the reagentresults in a failure of the system to follow the Cottrell curve decay,resulting in an inaccurate analyte concentration value being obtained.Thus, transient decay constants may be obtained from partially hydratedreagent layers resulting from relatively short initial incubationperiods.

Sensor strip reservoirs including a substantially constant concentrationdistribution of the measurable species may reduce any affect on thedecay constant attributable to c. The c term also may affect the decayconstant if the excitation duration is too long for the sample volume,resulting in a rapid decrease in the measurable species concentration asthe distance increases from the surface of the electrode. Using a shortexcitation or multiple short excitations combined with one or multiplerelaxations may assist in reducing the effect of the c term on the decayconstant.

For example, the '069 patent describes a system that provides a −0.5Cottrell decay constant when a 160 second initial incubation period iscombined with a 50 μL sample reservoir. For this system, if theincubation period were sufficiently shortened, the b term of Equation(2) would increase, thus providing non-Cottrell decay. Similarly, if thereservoir volume were sufficiently reduced, non-Cottrell decay wouldresult from an increase in the c term of Equation (2).

FIG. 5 depicts decay constants obtained from sensor strips havingreservoir volumes of about 3.5 μL and electrode to lid distances ofabout 250 μm after varying incubation periods for whole blood samplescontaining 50, 100, 200, or 400 mg/dL of glucose. The rate of decayincreased with increasing incubation time; however, a Cottrell decayconstant of −0.5 was not obtained within the six second incubationperiod. Thus, the system provided transient decays under thesecircumstances.

Table I, below, provides the decay constants for the 1-6 secondincubation periods of FIG. 5 and provides projected constants for 10 and15 second incubation periods. A projected decay constant also isprovided for an extended 20 second incubation period.

TABLE I Incu- Input bation Signal Period 50 mg/dL 100 mg/dL 200 mg/dL400 mg/dL 4-1-1 1 −0.2479 −0.23823 −0.2119 −0.17947 4-2-1 2 −0.337−0.30593 −0.282 −0.2631 4-4-1 4 −0.37417 −0.34993 −0.3442 −0.32837 4-5-15 −0.3877 −0.3734 −0.3549 −0.35283 4-6-1 6 −0.3979 −0.38273 −0.373−0.36483 Projec- 10 −0.44596 −0.42622 −0.42066 −0.42275 ted Projec- 15−0.4786 −0.45853 −0.45679 −0.46475 ted Projec- 20 −0.50176 −0.48146−0.48242 −0.49456 ted

In each instance, the input signal included an initial excitation offour seconds, followed by an open circuit type intermittent incubationperiod of varying duration, and a measurement excitation of one secondduring which the current was recorded. The sensor system did not achieveCottrell decay condition during any of the incubation periods from oneto six seconds. The sensor system would not be projected to achieve aCottrell decay condition within twelve seconds even at low 50 mg/dLglucose concentrations. Preferable transient decay constants are from−0.001 to −0.48 and from −0.52 to −1. More preferable transient decayconstants are at most −0.45, at most 0.35, and at most −0.3. Othertransient decay constants may be used.

FIGS. 6A-6C plot the current profiles obtained from three sensor stripseach having a different average initial thickness of the reaction layerat initial incubation periods of 0.125, 0.5, 1, 2, 4, and 6 seconds. Thesample reservoir of each strip was about 1 μL. The FIG. 6A plot wasobtained from multiple sensor strips having reaction layers with anaverage initial thickness from about 15 μm to about 20 μm (“thick”). TheFIG. 6B and 6 C plots were obtained from multiple sensor strips havingreaction layers with average initial thicknesses from 10 μm to 15 μm(“intermediate”) and from 1 μm to 2 μm (“thin”), respectively. Otherthicknesses may be used.

The figures establish the relationship of incubation time, reagent layerthickness, and the associated rate of layer hydration. Thicker reagentlayers required a longer time for the reagent layer to hydrate, and thegreater the time required for the reagent layer to hydrate, the longerthe time before the current decay reached a point of continual decrease.Current values obtained from decreasing transient decays are preferredfor correlating with the analyte concentration of the sample.

For the thick layered strips of FIG. 6A, continually decreasing currentdecays were obtained after an incubation period of about 4 seconds orgreater. However, for incubation periods of about 2 seconds and less, acontinually decreasing current decay was not obtained for thick layeredstrips until about 2 or more seconds of input signal were applied.

For the intermediate thickness reagent layer of the FIG. 6B sensorstrips, continually decreasing current decays were obtained after anincubation period of about 2 seconds or greater. For incubation periodsof about 1 second and less, about 2 or more seconds of input signalprovided a continually decreasing current decay.

For the thin reagent layer of the FIG. 6C sensor strips, continuallydecreasing current decays were obtained after an incubation period ofabout 1 second or greater. For incubation periods of about 0.5 secondand less, about 1 or more seconds of input signal provided a continuallydecreasing current decay. Thus, thinner reagent layers may be combinedwith shorter incubation periods to provide a shorter total analysistime, while thicker reagent layers may require longer durationincubation periods and/or input signals.

FIGS. 7A-7B plot the natural logs of current vs. time for whole bloodsamples including 100 or 300 mg/dL of glucose at 40% hematocrit obtainedafter a 6 second initial incubation period. The sample reservoir volumesand reagent layer initial average thicknesses were as in FIGS. 6A-6C,above. The plots were generated from current values obtained during thefirst 5 seconds of a 10 second excitation, where the a term of Equation(2) dominates the decay constant. Each of the observed decayconstants-slopes of the ln(current, nA) vs. ln(time, sec) plots-differfrom the −0.5 Cottrell decay constant, having transient decay constantsranging from about −0.35 to about −0.45. Thus, even at the longestinitial incubation period of 6 seconds, Cottrell decay is not observed.

FIGS. 8A-8C are current decay profiles from a 0.25 second initialincubation period followed by a gated input signal including 0.5 secondexcitations and 0.25 second relaxations, to provide a duty cycleduration of 0.75 second. Both intermediate and thin reagent layer sensorstrips having sample reservoir volumes of about 1 μL were used toanalyze whole blood samples including 50, 100, or 400 mg/dL of glucoseat 40% hematocrit. Continually decreasing current decays that may becorrelated to the 50 mg/dL analyte concentration in the sample wereobtained within 0.75 second for the thin reagent layer, thus during thefirst excitation. For the thicker intermediate reagent layer,continually decreasing current decays were obtained within 3 seconds,thus during the third excitation.

FIG. 8D is a calibration plot obtained by plotting the endpoint currents(p1, p2, p3) of the first three excitations obtained from thin reagentlayer sensor strips as depicted in FIGS. 8A-8C. The figure establishesthat current values taken after very short incubation periods of 0.25second in accord with the present invention may be accurately correlated(R²=0.999) with the actual plasma glucose concentration of whole bloodsamples.

FIG. 8E is a calibration plot obtained by plotting the endpoint currents(p4, p5, p6) of excitations 4, 5, and 6 obtained from sensor stripshaving intermediate thickness reagent layers as depicted in FIGS. 8A-8C.The figure establishes that current values taken after a very short 0.25second initial incubation period and multiple duty cycles including 0.5second excitations and 0.25 second relaxations in accord with thepresent invention may be accurately correlated (R²=0.99) with the actualplasma glucose concentration of whole blood samples.

To provide a clear and consistent understanding of the specification andclaims of this application, the following definitions are provided.

“Sample” is a composition that may contain an unknown amount of theanalyte. A sample may be aqueous, such as whole blood, urine, saliva, ora derivative, such as an extract, a dilution, a filtrate, or areconstituted precipitate.

“Incubation period” is the length of time that the sample reacts withthe reagents before an excitation is applied, such as before the firstexcitation is applied and/or the time between excitations if the inputsignal includes multiple excitations.

“Measurable species” is any electrochemically active species that may beoxidized or reduced under an appropriate potential at an electrodesurface.

An “Oxidoreductase” facilitates the oxidation or reduction of an analyteor biological substrate. See, for example the Oxford Dictionary ofBiochemistry and Molecular Biology, Revised Edition, A. D. Smith, Ed.,New York: Oxford University Press (1997) pp. 161, 476, 477, and 560.

While various embodiments of the invention have been described, it willbe apparent to those of ordinary skill in the art that other embodimentsand implementations are possible within the scope of the invention.

The invention claimed is:
 1. An electrochemical method for determiningan analyte concentration in a sample, comprising: receiving a sampleincluding a biological fluid at a sample reservoir, the sample reservoirhaving a reagent layer including an enzyme disposed in the samplereservoir and a pair of electrodes partially or completely disposed inthe sample reservoir such that the sample contacts both the enzyme andthe pair of electrodes responsive to being received at the samplereservoir, the contact between the sample and the enzyme causing areaction between the enzyme and an analyte in the sample such that theanalyte begins to undergo a redox conversion to electrochemically form ameasurable species from the reaction between the enzyme and the analyte,the electrochemical forming of the measurable species resulting from atransfer of electrons from or to the analyte during the redox conversionof the analyte; applying, by a signal generator, an electrical inputsignal having an excitation and a relaxation to the sample via the pairof electrodes after an incubation period, the incubation period beingless than a reference period, wherein at the end of the reference period(i) all of the analyte has undergone the redox conversion to themeasureable species and (ii) the measurable species has a constantconcentration distribution, and wherein at the end of the incubationperiod (i) a portion but not all of the analyte has undergone the redoxconversion to the measurable species and (ii) the measurable species hasa variant concentration distribution, the reference period being basedon a height of the sample reservoir, a volume of the sample reservoir, athickness of the reagent layer, or any combination thereof, theincubation period beginning when the sample is received at thereservoir, the applying of the electrical input signal producing anelectrical output signal that is based on the analyte concentration ofthe sample; measuring, by a processor, the electrical output signal; anddetermining, by the processor, the analyte concentration of the sampleas a function that includes the measured electrical output signal. 2.The method of claim 1, wherein the variant concentration distributionrepresents an incomplete redox conversion of the analyte to themeasurable species.
 3. The method of claim 1, wherein the variantconcentration distribution undergoes a substantial change from a surfaceof at least one of the pair of electrodes to an upper lid of the samplereservoir that is filled with the sample.
 4. The method of claim 3,wherein the height of the sample reservoir is from 100 to 250 μm.
 5. Themethod of claim 1, wherein the variant concentration distributionrepresents the measurable species undergoing a transient condition. 6.The method of claim 5, wherein the transient condition manifests as atransient decay of the output signal.
 7. The method of claim 6, whereinthe transient decay has a decay constant less than −0.5 or greater than−0.5.
 8. The method of claim 7, wherein the transient decay is anon-Cottrell decay.
 9. The method of claim 7, wherein the decay constantis from 0.52 to 1 or at most −0.35.
 10. The method of claim 1, whereinthe analyte concentration is determined before the analyte undergoescomplete conversion to the measurable species.
 11. The method of claim1, wherein the electrical input signal includes a plurality ofexcitations and a plurality of relaxations.
 12. The method of claim 11,wherein at least one of the excitations has a duration from 0.1 to 5seconds or from 0.1 to 1 second.
 13. The method of claim 11, wherein atleast one of the relaxations has a duration from 0.1 to 3 seconds. 14.The method of claim 1, wherein the electrical input signal is a voltageand the output signal is a current.
 15. The method of claim 1, whereinthe incubation period does not exceed 4 seconds.
 16. The method of claim1, wherein the incubation period does not exceed 1 second.
 17. Themethod of claim 1, wherein the incubation period does not exceed 0.1seconds.
 18. An electrochemical method for determining an analyteconcentration in a sample, comprising: receiving a sample including abiological fluid at a sample reservoir, the sample reservoir having areagent layer including an enzyme disposed in the sample reservoir and apair of electrodes partially or completely disposed in the samplereservoir such that the sample contacts both the enzyme and the pair ofelectrodes responsive to being received at the sample reservoir, thecontact between the sample and the enzyme causing a reaction between theenzyme and an analyte in the sample such that the analyte begins toundergo a redox conversion to electrochemically form a measurablespecies from the reaction between the enzyme and the analyte, theelectrochemical forming of the measurable species resulting from atransfer of electrons from or to the analyte during the redox conversionof the analyte; applying, by a signal generator, an electrical inputsignal having an excitation and a relaxation to the sample via the pairof electrodes after an incubation period, the incubation period beingless than a reference period, wherein at the end of the reference period(i) all of the analyte has undergone the redox conversion to themeasureable species and (ii) the measurable species has a constantconcentration distribution, and wherein at the end of the incubationperiod (i) a portion but not all of the analyte has undergone the redoxconversion to the measurable species and (ii) the measurable species hasa variant concentration distribution, the reference period being basedon a height of the sample reservoir, a volume of the sample reservoir, athickness of the reagent layer, or any combination thereof, theincubation period beginning when the sample is received at thereservoir, the applying of the electrical input signal producing anelectrical output signal that is based at least partially on the analyteconcentration of the sample and has a non-Cottrell transient decay;measuring, by a processor, the electrical output signal; anddetermining, by the processor, the analyte concentration of the sampleas a function that includes the measured electrical output signal.
 19. Amethod for determining an analyte concentration in a sample, comprising:receiving a sample including a biological fluid at a sample reservoir,the sample reservoir having a reagent layer including an enzyme disposedin the sample reservoir and a pair of electrodes partially or completelydisposed in the sample reservoir such that the sample contacts both theenzyme and the pair of electrodes responsive to being received at thesample reservoir, the contact between the sample and the enzyme causinga reaction between the enzyme and an analyte in the sample such that theanalyte begins to undergo a redox conversion to electrochemically form ameasurable species from the reaction between the enzyme and the analyte,the electrochemical forming of the measurable species resulting from atransfer of electrons from or to the analyte during the redox conversionof the analyte; applying, by a signal generator, an electrical inputsignal having an excitation and a relaxation to the sample via the pairof electrodes after an incubation period, the incubation period beingless than a reference period, wherein at the end of the reference period(i) all of the analyte has undergone the redox conversion to themeasureable species and (ii) the measurable species has a constantconcentration distribution, and wherein at the end of the incubationperiod (i) a portion but not all of the analyte has undergone the redoxconversion to the measurable species and (ii) the measurable species hasa variant concentration distribution, the reference period being basedon a height of the sample reservoir, a volume of the sample reservoir, athickness of the reagent layer, or any combination thereof, theincubation period beginning when the sample is received at thereservoir, the applying of the electrical input signal producing anelectrical output signal that is based at least partially on the analyteconcentration of the sample and has a decay constant less than −0.5 orgreater than −0.5; measuring, by a processor, the electrical outputsignal; and determining, by the processor, the analyte concentration ofthe sample as a function that includes the measured electrical outputsignal.
 20. The method of claim 1, wherein the output signal is producedfrom the excitation of the electrical input signal.